BROOKHAVEN SCIENCE ASSOCIATESFunded under contract: DE-AC02-98CH10886

U.S. DEPARTMENT OF ENERGY

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Infrared Imaging and MicrospectroscopyInfrared Imaging and Microspectroscopy

Lisa M. MillerNational Synchrotron Light SourceBrookhaven National Laboratory

Upton, NY 11973

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Chemical Features of Biological ComponentsChemical Features of Biological Components

Protein O-NH-C-CH-

=

R

Nucleic Acid

O=P-O

O

Lipid OCH3-(CH2)n-C-OH

=

Carbohydrate

Abs

orba

nce

1000 2000 3000 Frequency (cm-1)

Amide IAmide II

CH stretch C=O esterstretch

P=O stretch

C-O stretchOH stretch

protein

lipid

nucleicacid

carbohydrate

C-Cstretch

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Protein Structure Determination with FTIRProtein Structure Determination with FTIR

α-helix

β-sheetβ-turn

extended coil

Amide I Secondary Structure Assignments:

1620 - 1640 β-sheet1645 extended coil (D2O)1648 - 1657 α-helix1660 triple helix1670 - 1695 β-sheet, β-turn

Collagen(triple helix)

Myoglobin(α-helix)

Abeta Amyloid(α-helix + β-sheet)

Cytochrome c(α-helix + coil)

STI(β-sheet + coil)

1600 1650 1700 Frequency (cm-1)

A

B

C

D

E

A

B

C

D

E

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IR Spectroscopy vs. Micro-SpectroscopyIR Spectroscopy vs. Micro-Spectroscopy

Bulk samplesKBr

solid+

Spatial Resolution:~1-3 mm

+solution

Microscopic Heterogeneity

Spatial Resolution:~ 25-30 µm (globar)~ 5-10 µm (synchrotron)

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Why SYNCHROTRON Infrared Microspectroscopy?Why SYNCHROTRON Infrared Microspectroscopy?

100 µm 20 µm

Biological sample are SMALL:• spatially• in concentration

BRIGHTNESS (1000x)

Higher Spatial Resolution

Better Signal-to-Noise

Faster Data Collection

Spectroscopy

+

BROADBAND

Synchrotron Advantages:

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Single Red Blood Cell: Globar vs. Synchrotron SourceSingle Red Blood Cell: Globar vs. Synchrotron Source

Diffraction Limit4000 cm-1 2.5 µm2950 cm-1 3.4 µm1650 cm-1 6.1 µm1200 cm-1 8.3 µm1000 cm-1 10 µm600 cm-1 17 µm200 cm-1 50 µm

• Synchrotron IRMS is diffraction-limited

• Conventional IRMS is throughput-limited

10 µm

1000 2000 3000 Frequency (cm-1)

5x5 µm

synchrotron

globar

P/P noise:0.0038

0.0575

0 10 20 30 40 50 60 700

4

8

12

16

MC

T-A

* Sig

nal (

V)

Aperture size (µm)

synchrotron

globar

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Beamline U10BBeamline U10B

Synchrotron infrared beam pipe

Nicolet Magna 860 FTIR

Spectra Tech ContinuµmIR microscope

Filter cube turret

UV (quartz) light source

Schwarzchild 32x IR objective

Automated mapping stage

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Chemical Content of New Bone in OsteoporosisChemical Content of New Bone in Osteoporosis

Normal illumination

Abso

rban

ce

0.5 OD

(1)

(2)

(3)

1800 1400 1000 600Frequency (cm-1)

(4)

(5)

(6)ALIZARIN(t=2 yr.)

CALCEIN (t=1 yr.)

Fluorescence illumination

• Rate of bone mineral formation is slower in osteoporosis

L.M. Miller, J. Tibrewala, C.S. Carlson. Cell. Mol. Biol., 46:1035-44 (2000).

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Prion Protein Structure and LocationPrion Protein Structure and Location

1620 1660 1700

10 µm

Frequency (cm-1)

• Hamster scrapie 263K• Dorsal root ganglia• Terminally infected

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Chemical Imaging of HairChemical Imaging of Hair

PhospholipidsPhospholipidsPhospholipidscuticle (3-5 µm)

cortex (40-100 µm)

medulla (5-10 µm)

ProteinsProteinsProteins

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Protein Aggregate Formation in Alzheimer’s PlaquesProtein Aggregate Formation in Alzheimer’s Plaques

• misfolded, aggregated protein is associated with Abeta plaques 2001000 300

200

100

0

Microns

0.0

1.0

L.M. Miller, P. Dumas, N. Jamin, J.-L. Teillaud, J. Miklossy, L. Forro (2002). Rev. Sci. Instr., 73: 1357-60.

0.00

0.10

0.20

0.30

0.40

0.50

Log

(1/R

)

1400 1500 1600 1700 1800 Frequency (cm-1)

NORMAL

MISFOLDED

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Metal Accumulation in Alzheimer’s PlaquesMetal Accumulation in Alzheimer’s Plaques

Cu Kα

Zn Kα

• Zn and Cu are associated with Abeta plaques

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Correlation Between Metal Accumulation and Protein AggregationCorrelation Between Metal Accumulation and Protein Aggregation

Zn

Abeta

misfolded protein

Zn + Abeta

Abeta + misfolded protein

Zn + misfolded protein

Metal

Thioflavin SFluorescence

MisfoldedProtein

composite

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Chemical Imaging of Mitotic CellsChemical Imaging of Mitotic Cells

Max

Min-10 0 10 20 300 5 10 15 20 25 30 350

5

10

15

20PROTEIN (1650 cm-1)

-10 0 10 20 300 5 10 15 20 25 30 350

5

10

15

20LIPIDS (2925 cm-1) Max

Min

OPTICAL IMAGE

10 µm 10 µm

N. Jamin, J.L. Teillaud, P. Dumas, G.L. Carr, G.P. Williams (1998). PNAS 95: 4837-40.

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Chemical Changes in Apoptotic CellsChemical Changes in Apoptotic Cells

ell

ptosis l)

ptosis ell)

108512

40

1541

1651

1043

1175

1223

1455

1539

1655

1043

1175

1223

1455

154316

55

1000 3000 Frequency (cm-1)

2000

Abs

orba

nce

20 µm

20 µm

1175-1180 cm-1

5 µm

How does apoptosis proceed in real time?

L.M. Miller, P. Dumas, N. Jamin, J.-L. Teillaud, J. Miklossy, L. Forro (2002). Rev. Sci. Instr., 73: 1357-60.

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Impact of NSLS-II: Schematic Brightness ComparisonImpact of NSLS-II: Schematic Brightness Comparison

Frequency/Photon Energy (log scale)

Brig

htne

ss (

log

scal

e)

NSLS-II (500ma, 20mr)

VUV (700ma)

40m

r

90m

r

Mid-IRFar-IRTHz

400

cm-1, 2

5 µm

, 50

meV

4000

cm

-1, 2

.5 µ

m, 0

.5 e

V• Mid IR: NSLS-II will have equal brightness to NSLS • Far-IR and multi-point imaging: disadvantaged by NSLS-II

X-ray

Infrared

Opening Angle

IR flux is primarily dependent upon ring current in mid-IR

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SummarySummaryOverview

• Infrared microspectroscopy and imaging can be used to image the chemical makeup of biological tissues and cells

• The spatial resolution of synchrotron IRMS is diffraction-limited at 2-20 µm

Importance of Research• Chemical imaging of mineralized tissues, in situ protein, lipid, nucleic acid

content, protein structure

Scientific Challenges and Opportunities• Multi-technique imaging: combining x-ray, visible, IR imaging techniques• Improve spatial resolution• Improve data collection rates

Impact of NSLS-II• NSLS-II brightness will slightly improve the mid-IR imaging capabilities• NSLS-II will prevent the creation of large opening-angle beamlines for (1) far-IR

applications, and (2) line-source (i.e. multi-point) imaging

Synergy and Demand• Combination of IRMS with x-ray imaging techniques• Currently 4 IRMS bending magnet beamlines; need at least this many for NSLS-II